1. Field of the Invention
The present invention relates to activation of neurotrophin receptors and a screening method for molecules that activate neurotrophin receptors in the absence of neurotrophins.
2. Description of the Related Art
Neurotrophins play a prominent role in the development of the vertebrate nervous system by influencing cell survival, differentiation and cell death events (Levi-Montalcini, 1987; Lewin et al, 1996). Neurotrophins also exhibit acute regulatory effects upon neurotransmitter release, synaptic strength and connectivity (Thoenen, 1996; Bonhoeffer, 1996). In addition to promoting axonal and dendritic branching, neurotrophins serve as chemoattractants for extending growth cones in vitro (Gallo et al, 1997). These actions are mediated by neurotrophin binding to two separate receptor classes, the Trk family of tyrosine kinase receptors and the p75 neurotrophin receptor, a member of the TNF receptor superfamily (Chao and Hempstead, 1995). Binding of neurotrophins to Trk receptors results in receptor autophosphorylation and downstream phosphorylation cascades.
Mutations in Trk neurotrophin receptor function lead to deficits in survival, axonal and dendritic branching, long term potentiation and behavior (McAllister et al, 1999; Minichiello et al, 1999; Lyons et al, 1999). NGF, BDNF, NT-3 and NT-4 also bind to the p75 neurotrophin receptor, a potential cell death receptor whose actions are negated by Trk tyrosine kinase signaling (Dobrowsky et al, 1995; Yoon et al, 1998). Therefore, the ability to regulate Trk tyrosine kinase activity is critical for neuronal survival and differentiation.
Neurotrophic factors exemplified by the neurotrophins (NGF, BDNF, NT-3 and NT-4/5), ciliary neurotrophic factor (CNTF) and glial derived neurotrophic factor (GDNF) all utilize intracellular tyrosine phosphorylation to mediate neuronal cell survival (Segal and Greenberg, 1996; Kaplan and Miller, 2000). CNTF acts through a complex of gp130, CNTF receptor and LIF subunits which are linked to JAK/STAT signaling molecules, whereas the GDNF receptor consists of the c-Ret receptor tyrosine kinase and a separate α-binding protein. Actions of the NGF family of neurotrophins are dictated by the Trk family of receptor tyrosine kinases and the p75 receptor, a member of the TNF receptor superfamily. The neurotrophins have been under investigation for some time as therapeutic agents for the treatment of neurodegenerative diseases and nerve injury, such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease), Parkinson's disease, peripheral neuropathy and spinal cord injury, either individually or in combination with other trophic factors such as CNTF.
The first clinical trials using neurotrophic factors have led to failures (Verrall, 1994; ALS CNTF Treatment Study Group, 1996; Miller et al., 1996; Sendtner, 1997; BDNF Study Group, 1999). Although there is abundant evidence that neurotrophic factors provide neuroprotection in a great variety of experimental systems (Hefti, 1997), the therapeutic procedures for delivering these proteins to patients have not been effective. Subcutaneous administration of CNTF and BDNF in ALS patients was unsuccessful because these proteins did not reach the motor neurons of the spinal cord and brain stem. Systemic treatment of these proteins failed to reach the therapeutic target. Another serious problem was gauging the optimal dosages of neurotrophic factors. Under supramaximal concentrations of BDNF, desensitization or a limitation of the supportive actions of BDNF resulted (Vejsada et al., 1994). This was likely due to downregulation of TrkB receptors. Neurotrophins may also lead to opposite effects on neuronal survival and regrowth of axons over long distances (Thoenen, 2001).
Another approach is to deliver these molecules directly into the brain. This procedure overcomes the problems of systemic administration due to the blood-brain barrier and reaching populations of neurons in the central nervous system that do not project to the periphery. This approach also presents logistic problems. A small number of Alzheimer's patients in Sweden have received intraventricular NGF infusion using pumps, based upon rodent studies in which cognitive deficits in rats could be improved with NGF treatment (Fischer et al., 1987). However, several acute side reactions occurred in these patients from NGF infusion, including pronounced pain, that prevented a meaningful assessment of efficacy (Johagen et al., 1998). Many side effects, including weight loss, diarrhea, hyperplasia, increased epileptic and motor activity, have been documented when high levels of neurotrophins have been administered in animal models or in human trials of ALS (ALS CNTF Treatment Study Group, 1996; Kobayashi et al., 1997; Winkler et al., 1997; BDNF Study Group, 1999; Thoenen, 2001). Besides the problems in managing the dose and pharmacokinetics of these proteins in the nervous system, there is also abundant evidence that neurotrophins can cause apoptosis in the nervous system (Rabizadeh et al., 1993; Casaccia-Bonnefil et al., 1998) These observations demonstrate the limitations of intraventricular and intracerebral infusion of neurotrophic factors as therapeutic intervention for neurodegenerative diseases.
G protein-coupled receptors (GPCR) mediate transmembrane signaling for a large number of ligands, including hormones, neurotransmitters, photons, odorants, pheromones and chemokines. These receptors relay signals to heterotrimeric G proteins which directly modulate the activity of enzymes and ion channels. Every receptor has a similar topology with seven membrane-spanning domains and shares an ability to act through a common signaling mechanism. When activated, a receptor associates with guanine nucleotide regulatory proteins, or G proteins. G proteins are associated with the membrane and consist of three subunits, α, β and γ. The G proteins serve to amplify receptor signaling by exchanging GTP for GDP bound to Gα, followed by the dissociation of Gβ and the Gγ subunits from the receptor. Free Gα couples to effector enzymes, such as adenylate cyclase, guanylate cyclase and phospholipases. A number of second messengers, such as diacylglycerol, IP3, cAMP and cGMP are produced and can influence ion channel activities, such as Ca+2 and K+channels (Gudermann et al., 1997).
Many GPCRs are capable of activating the mitogen-activated protein (MAP) kinase signaling pathway, in addition to downstream effector enzymes such as adenylyl cyclase and phospholipase C (Dhanasekaran et al., 1995; van Biesen et al., 1996; Gudermann, 2001). These events result in increased cell division and growth. GPCR signaling is a complex system that involves regulatory feedback desensitization and protein phosphorylation events. The receptors in this superfamily are diverse at the amino acid sequence level and in their functional responses. The natural ligands of different GPCR members range from non-peptide neurotransmitters to odors and light. Other ligands include lipids such as lysophosphatic acid (LPA); eicosanoids such as prostaglandins; amino acids and ions such as glutamate and calcium; peptides and proteins, such as angiotensin, bradykinin and thrombin; and biogenic amines such as acetylcholine, serotonin and melatonin.
While the signaling cascades initiated by GPCRs cause a large number of metabolic responses and give changes in gene expression leading to cell proliferation and differentiation, little attention has been given to their possible involvement in neuronal survival events. For example, induction of mitogenic events has been observed through signaling from several G protein-coupled receptors that result in an increase in receptor tyrosine kinase phosphorylation (Daub et al., 1996 and Luttrell et al., 1999). Transactivation of EGF and PDGF receptors occurs with LPA, thrombin and carbacol, but the functional consequences of this signaling has not been determined. Whether transactivation of neurotrophic factor receptor tyrosine kinases occurs via G protein-coupled receptors has not been demonstrated to date.
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